M30 CytoDeath™ CK18 Kit

The M30 CytoDeath™ ELISA is a novel research kit developed for cell culture applications. The M30 CytoDeath™ assay has a dynamic range and sensitivity suitable for in vitro work.

M30 CytoDeath™ ELISA is a powerful drug screening tool. It is useful for in vitro characterization of apoptosis-inducing drugs, including establishment of time course kinetics and dose response relationships. The effects of large numbers of siRNAs on apoptosis can be tested in the 96 well format. The assay offers a unique possibility to quantify apoptosis of epithelially derived cells in multicellular spheroids and organ culture systems.

The M30 CytoDeath™ ELISA is based on the M30 antibody (detecting the Asp396 caspase-cleavage site on CK18) and antibody M6. This combination is different from that used in the M30-Apoptosense® ELISA.

M6 Coated Microstrips: One microplate, 12 strips with 8 wells each, 96 dry wells in total. The wells are coated with mouse monoclonal K18 antibody M6. The microplate is sealed in an aluminium bag, which contains a desiccating device. If not all the strips are used, reseal the bag and keep the desiccating device inside. Ready for use!

The M30 CytoDeath™ ELISA is a solid-phase sandwich enzyme immunoassay. Standards and samples react with a solid phase capture antibody M6 directed against K18 and the HRP (horseradish peroxidase) conjugated M30 antibody directed against the K18Asp396 neo-epitope. Unbound conjugate is removed by a washing step. TMB Substrate is added. The color development is stopped and the absorbance is read. The resulting color is directly proportional to the concentration of the analyte. By plotting a standard curve from known concentrations versus measured absorbance, the amount of antigen in the sample can be calculated. The concentration of the antigen is expressed as units per liter (U/L).

In a study presented at the SOT Meeting in 2014, toxicity testing on cryopreserved differentiated HepaRG™ cells was performed using CK18 kits. HepaRG™ cells exhibit many characteristics of primary human hepatocytes, including morphology and expression of key metabolic enzymes, nuclear receptors, and drug transporters. Unlike HepG2 cells, HepaRG™ cells have high P450 activity and complete expression of all nuclear receptors. HepaRG™ was exposed to different compounds, including paracetamol, chloropromazine, rotenone, rosiglitazone and omeprazole. Toxicity and apoptosis were assayed from collected cell culture supernatants measuring M65 and M30 levels. M65 results were consistent with cell viability, whereas, M30 results indicated that apoptosis was induced at lower drug concentrations while necrosis was more prominent at higher ones (see figure below).

M65 and M30 kits robustly detect cell toxicity and apoptosis in HepaRG cells in vitro experiments. A additional key feature is that the CK18 kits measure an accumulated caspase-derived product, so there is no risk to miss time of apoptosis and transient caspase activation (See figure below).